Prophylactic and therapeutic treatment of infectious and other diseases with mono-and disaccharide-based compounds

ABSTRACT

Methods and compositions for treating or ameliorating diseases and other conditions, such as infectious diseases, autoimmune diseases and allergies are provided. The methods employ mono- and disaccharide-based compounds for selectively stimulating immune responses in animals and plants.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 09/861,466, filed, May 18, 2001, which isincorporated in its entirety herein.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

[0002] NOT APPLICABLE

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK.

[0003] NOT APPLICABLE

BACKGROUND OF THE INVENTION

[0004] The innate immune system coordinates the inflammatory response topathogens by a system that discriminates between self and non-self viareceptors that identify classes of molecules synthesized exclusively bymicrobes. These classes are sometimes referred to as pathogen associatedmolecular patterns (PAMPs) and include, for example, lipopolysaccharide(LPS), peptidoglycans, lipotechoic acids, and bacterial lipoproteins(BLPs).

[0005] LPS is an abundant outer cell-wall constituent from gram-negativebacteria that is recognized by the innate immune system. Although thechemical structure of LPS has been known for some time, the molecularbasis of recognition of LPS by serum proteins and/or cells has onlyrecently begun to be elucidated. In a series of recent reports, a familyof receptors, referred to as Toll-like receptors (TLRs), have beenlinked to the potent innate immune response to LPS and other microbialcomponents. All members of the TLR family are membrane proteins having asingle transmembrane domain. The cytoplasmic domains are approximately200 amino acids and share similarity with the cytoplasmic domain of theIL-1 receptor. The extracellular domains of the Toll family of proteinsare relatively large (about 550-980 amino acids) and may containmultiple ligand-binding sites.

[0006] The importance of TLRs in the immune response to LPS has beenspecifically demonstrated for at least two Toll-like receptors, Tlr2 andTlr4. For example, transfection studies with embryonic kidney cellsrevealed that human Tlr2 was sufficient to confer responsiveness to LPS(Yang et al., Nature 395:284-288 (1998); Kirschning et al. J Exp Med.11:2091-97 (1998)). A strong response by LPS appeared to require boththe LPS-binding protein (LBP) and CD14, which binds LPS with highaffinity. Direct binding of LPS to Tlr2 was observed at a relatively lowaffinity, suggesting that accessory proteins may facilitate bindingand/or activation of Tlr2 by LPS in vivo.

[0007] The importance of Tlr4 in the immune response to LPS wasdemonstrated in conjunction with positional cloning in lps mutant mousestrains. Two mutant alleles of the mouse lps gene have been identified,a semidominant allele that arose in the C3H/HeJ strain and a second,recessive allele that is present in the C57BL/10ScN and C57BL/10ScCrstrains. Mice that are homozygous for mutant alleles of lps aresensitive to infection by Gram-negative bacteria and are resistant toLPS-induced septic shock. The lps locus from these strains was clonedand it was demonstrated that the mutations altered the mouse Tlr4 genein both instances (Portorak et al., Science 282:2085-2088 (1998);Qureshi et al., J Exp Med 4:615-625 (1999)). It was concluded from thesereports that Tlr4 was required for a response to LPS.

[0008] The biologically active endotoxic sub-structural moiety of LPS islipid-A, a phosphorylated, multiply fatty-acid-acylated glucosaminedisaccharide that serves to anchor the entire structure in the outermembrane of Gram-negative bacteria. We previously reported that thetoxic effects of lipid A could be ameliorated by selective chemicalmodification of lipid A to produce monophosphoryl lipid A compounds(MPL□ immunostimulant; Corixa Corporation; Seattle, Wash.). Methods ofmaking and using MPL□ immunostimulant and structurally like compounds invaccine adjuvant and other applications have been described (see, forexample, U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094;4,987,237; Johnson et al., J Med Chem 42:4640-4649 (1999); Ulrich andMyers, in Vaccine Design: The Subunit and Adjuvant Approach; Powell andNewman, Eds.; Plenum: New York, 495-524, 1995; the disclosures of whichare incorporated herein by reference in their entireties). Inparticular, these and other references demonstrated that MPL□immunostimulant and related compounds had significant adjuvantactivities when used in vaccine formulations with protein andcarbohydrate antigens for enhancing humoral and/or cell-mediatedimmunity to the antigens.

[0009] Moreover, we have previously described a class of synthetic mono-and disaccharide mimetics of monophosphoryl lipid A, referred to asaminalkyl glucosaminide phosphates (AGPs), for example in U.S. Ser. No.08/853,826, now U.S. Pat. Nos. 6,113,918, 09/074,720, 09/439,839, nowU.S. Pat. No. 6,303,347, and in PCT/US98/09385 (WO 98/50399, Oct. 12,1998) the disclosures of which are incorporated herein by reference intheir entireties. Like monophosphoryl lipid A, these compounds have beendemonstrated to retain significant adjuvant characteristics whenformulated with antigens in vaccine compositions and, in addition, havesimilar or improved toxicity profiles when compared with monophosphoryllipid A. A significant advantage offered by the AGPs is that they arereadily producible on a commercial scale by synthetic means.

[0010] Although monophosphoryl lipid A and the AGPs have been describedprimarily for use in combination with antigens in vaccine formulations,their use as monotherapies, in the absence of antigen, for theprophyhlactic and/or therapeutic treatment of plant and animal diseasesand conditions, such as infectious disease, autoimmunity and allergies,has not been previously reported.

[0011] The present invention, as a result of a growing understanding ofcertain mechanisms underlying the activities of monophosphoryl lipid Aand AGP compounds, makes possible the novel therapeutic opportunitiesdescribed herein.

BRIEF SUMMARY OF THE INVENTION

[0012] In one aspect, the present invention provides methods fortreating, ameliorating or substantially preventing a disease orcondition in an animal by administering an effective amount of acompound having the formula:

[0013] and pharmaceutically acceptable salts thereof, wherein X is —O—or —NH—; R¹ and R² are each independently a (C₂-C₂₄)acyl group,including saturated, unsaturated and branched acyl groups; R³ is —H or—PO₃R¹¹R¹², wherein R¹¹ and R¹² are each independently —H or(C₁-C₄)alkyl; R⁴ is —H, —CH₃ or —PO₃R¹³R¹⁴, wherein R¹³ and R¹⁴ are eachindependently selected from —H and (C₁-C₄)alkyl; and Y is a radicalselected from the formulae:

[0014] wherein the subscripts n, m, p and q are each independently aninteger of from 0 to 6; R⁵ is a (C₂-C₂₄)acyl group (including, as above,saturated, unsaturated and branched acyl groups); R⁶ and R⁷ areindependently selected from H and CH₃; R⁸ and R⁹ are independentlyselected from H, OH, (C₁-C₄)alkoxy, —PO₃H₂, —OPO₃H₂, —SO₃H, —OSO₃H,—NR¹⁵R¹⁶, —SR¹⁵, —CN —NO₂, —CHO, —CO₂R¹⁵, —CONR¹⁵R¹⁶, —PO₃R¹⁵R¹⁶,—OPO₃R¹⁵R¹⁶, —SO₃R¹⁵ and —OSO₃R¹⁵, wherein R¹⁵ and R¹⁶ are eachindependently selected from H and (C₁-C₄)alkyl; R¹⁰ is selected from H,CH₃, —PO₃H₂, ω-phosphonooxy(C₂-C₂₄)alkyl, and ω-carboxy(C₁-C₂₄)alkyl;and Z is —O— or —S—; with the proviso that when R³ is —PO₃R¹¹R¹², R⁴ isother than —PO₃R¹³R¹⁴.

[0015] In certain illustrative aspects of the invention, the abovemethods are employed in treating, ameliorating or substantiallypreventing infectious diseases, autoimmune diseases and allergies.

[0016] The present invention, in other aspects, provides pharmaceuticalcompositions comprising one or more of the compounds described above ina suitable excipient, formulated and/or administered in the absence ofexogenous antigen.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 is a graph depicting the nonspecific protection of miceagainst lethal influenza challenge following or coincident withMonoPhosphoryl Lipid A (MPL) administration.

[0018]FIG. 2 is a graph depicting clinical symptoms following intranasaladministration of L-Seryl Aminoalkyl Glucosaminide Phosphates (AGPs) tomice.

[0019]FIG. 3 is a graph depicting clinical symptoms following L-SerylAminoalkyl Glucosaminide Phosphates (AGPs) monotherapy and influenzachallenge.

[0020] FIGS. 4-6 are graphs depicting cytokine induction by RC522 ascompared to MPL in overnight whole blood cultures from three humandonors (donors A-C, respectively).

[0021]FIG. 7 are graphs depicting cytokine induction by RC522 ascompared to MPL in short term whole blood cultures from donor A.

[0022]FIG. 8 are graphs depicting cytokine induction by RC522 ascompared to MPL in murine (Balb/c and C3H/HEJ) splenic cultures.

[0023]FIG. 9 are graphs depicting cytokine induction by RC529 and RC552as compared to MPL in human peripheral blood mononuclear cells (PBMC).

[0024] FIGS. 10 A-E is a figure showing various AGP compounds.

[0025]FIG. 11 is a graph showing clinical symptoms following intranasaladministration of L-Seryl Aminoalkyl Glucosaminide Phosphatesmonotherapy and influenza challenge.

[0026]FIG. 12 is a graph showing clinical symptoms following intranasaladministration of L-Seryl 666 versus L-Seryl 000 AminoalkylGlucosaminide Phosphates monotherapy and influenza challenge.

[0027]FIG. 13 is a graph showing clinical symptoms following intranasaladministration of L-Seryl Aminoalkyl Glucosaminide Phosphatesmonotherapy and influenza challenge. L-Seryl compounds have variouscombinations of 6 and 10 carbon fatty acid chains in the secondary fattyacid position.

[0028]FIG. 14 is a graph showing clinical symptoms following intravenousadministration of L-Seryl Aminoalkyl Glucosaminide Phosphatesmonotherapy and intravenous Listeria monocytogenes challenge.

[0029]FIG. 15 is a graph showing clinical symptoms following intravenousadministration of L-Seryl Aminoalkyl Glucosaminide Phosphatesmonotherapy and intravenous Listeria monocytogenes challenge. L-Serylcompounds have various combinations of 6 and 10 carbon fatty acid chainsin the secondary fatty acid position.

[0030]FIG. 16 is a graph showing clinical symptoms following intravenousadministration of various Aminoalkyl Glucosaminide Phosphatesmonotherapy and intravenous Listeria monocytogenes challenge. The AGPcompounds all have 10 carbon fatty acid chains in the secondary fattyacid position.

[0031]FIG. 17 is a graph showing clinical symptoms following intravenousadministration of Aminoalkyl Glucosaminide Phosphates monotherapy andintravenous Listeria monocytogenes challenge. The AGP compounds vary inlinker length between the glucosamine and aglycone moieties.

DETAILED DESCRIPTION OF THE INVENTION

[0032] Illustrative Prophylactic and Therapeutic Applications

[0033] The present invention broadly concerns prophylactic andtherapeutic methods of treating certain diseases and other medicalconditions by administration of an effective amount of one or more mono-or disaccharide compounds described herein or a pharmaceuticalcomposition comprising one or more such compounds. While certain of themono- and disaccharide compounds have been described for use asadjuvants in combination with exogenously administered antigens invaccine formulations, and for use in certain other applications, thepresent invention provides novel therapeutic methods that employ thecompounds preferably in monotherapeutic applications, i.e., in theabsence of exogenously administered antigen.

[0034] Thus, in one aspect, the present invention provides methods fortreating, ameliorating and/or substantially preventing infectiousdiseases in eukaryotic subjects, particularly in animals, preferably inhumans. Given the importance of TLR-mediated signalling in the innateimmune response to microbial challenge, the ability to stimulate suchpathways selectively and with minimal toxicity represents a powerfulapproach for prophylactic and/or therapeutic treatment modalitiesagainst a wide range of infectious agents.

[0035] The methods described herein are applicable against essentiallyany type of infectious agent, including bacteria, viruses, parasites,and fungi. Illustratively, the invention is useful for the prophylacticand/or therapeutic treatment of bacterial infections by species fromPseudomonas, Escherichia, Klebsiella, Enterobacter, Proteus, Serratia,Candida, Staphylococci, Streptococci, Chlamydia, Mycoplasma and numerousothers. Illustrative viral conditions that may be treated in accordancewith the invention include those caused, for example, by Influenzaviruses, Adenoviruses, parainfluenza viruses, Rhinoviruses, respiratorysyncytial viruses (RSVs), Herpes viruses, Cytomegaloviruses, Hepatitisviruses, e.g., Hepatitis B and C viruses, and others. Illustrative fungiinclude, for example, Aspergillis, Candida albicans, Cryptococcusneoformans, Coccidioides immitus, and others.

[0036] In one illustrative embodiment, the invention provides methodsfor the treatment of subjects, particularly immunocompromised subjects,that have developed or are at risk for developing infections, such asnosocomial bacterial and viral infections. About 2 million of the 40million individuals hospitalized every year develop nosocomial infectionduring their stay and about 1% of these, or about 400,000 patients,develop nosocomial pneumonia, more than 7000 of which die. This makesnosocomial pneumonia the leading cause of death in hospital-acquiredinfections. Thus, this embodiment fills a significant need for effectiveprophylactic approaches in the treatment of nosocomial infections.

[0037] In a related embodiment, the present invention providesprophylactic treatments for immunocompromised patients, such asHIV-positive patients, who have developed or are at risk for developingpneumonia from either an opportunistic infection or from thereactivation of a suppressed or latent infection. In 1992, about 20,000cases of Pneumocystis carinii infections in AIDS patients were reportedin the U.S. alone. Additionally, 60-70% of all AIDS patients getP.carinii at some time during their illness. Thus, the present inventionin this embodiment provides effective prophylactic methods for thisat-risk population.

[0038] In another related embodiment, the methods of the presentinvention are used for treating other patient populations that may beimmunocompromised and/or at risk for developing infectious diseases,including, for example, patients with cystic fibrosis, chronicobstructive pulmonary disease and other immunocompromized and/orinstitutionalized patients.

[0039] In support of these and other embodiments of the invention, wehave demonstrated that pre-challenge administration of an illustrativecompound of the present invention in immunocompromised mice providessignificant prophylactic protection against infection by Pneumocystiscarinii. (See Example 1).

[0040] In another aspect of the invention, the mono- and disaccharidecompounds described herein are employed in methods for treating,ameliorating or substantially preventing allergic disorders andconditions, such as sinusitis, chronic rhinosinusitus, asthma, atopicdermatitis and psoriasis. This approach is based at least in part on theability of the mono- and disaccharide compounds to activate theproduction of cytokines from target cells that can compete withstereotypic allergic-type cytokine responses characterized by IL-4production or hyperresponsiveness to IL-4 activity. Administration ofcertain of the mono- and disaccharide compounds disclosed herein resultsin IFN-gamma and IL-12 expression from antigen processing and presentingcells, as well as other cells, resulting in down regulation of cytokinesassociated with allergic responses such as IL-4, 5, 6, 10 and 13.

[0041] In another aspect of the invention, mono- and disaccharidecompounds are employed in methods for treating autoimmune diseases andconditions. The mono- and disaccharide compounds for use in thisembodiment will typically be selected from those capable ofantagonizing, inhibiting or otherwise negatively modulating one or moreToll-like receptors, particularly Tlr2 and/or Tlr4, such that anautoimmune response associated with a given condition is ameliorated orsubstantially prevented. Illustratively, the methods provided by thisembodiment can be used in the treatment of conditions such asinflammatory bowel disease, rheumatoid arthritis, chronic arthritis,multiple sclerosis and psoriasis.

[0042] While not wishing to be bound by theory, it is believed that theefficacy of the prophylactic and therapeutic applications describedabove are based at least in part on the involvement of the mono- anddisaccharide compounds in the modulation of Toll-like receptor activity.In particular, Toll-like receptors Tfr2, Tlr4, and others, are believedto be specifically activated, competitively inhibited or otherwiseaffected by the non-toxic LPS derivatives and mimetics disclosed herein.Accordingly, the methods of the invention provide a powerful andselective approach for modulating the innate immune response pathways inanimals without giving rise to the toxicities often associated with thenative bacterial components that normally stimulate those pathways.

[0043] Illustrative Mono- and Disaccharide Compounds

[0044] Illustrative mono- or disaccharide compounds employed in theabove prophylactic and therapeutic applications comprise compoundshaving the formula:

[0045] and pharmaceutically acceptable salts thereof, wherein X is —O—or —NH—; R¹ and R² are each independently a (C₂-C₂₄)acyl group,including saturated, unsaturated and branched acyl groups; R³ is —H or—PO₃R¹¹R¹², wherein R¹¹ and R¹² are each independently —H or (C₁C₄)alkyl; R⁴ is —H, ═CH₃ or —PO3R¹³R¹⁴, wherein R¹³ and R¹⁴ are eachindependently selected from —H and (C₁ C₄)alkyl; and Y is a radicalselected from the formulae:

[0046] wherein the subscripts n, m, p and q are each independently aninteger of from 0 to 6; R⁵ is a (C₂-C₂₄)acyl group (including, as above,saturated, unsaturated and branched acyl groups); R⁶ and R⁷ areindependently selected from H and CH₃; R⁸ and R⁹ are independentlyselected from H, OH, (C₁-C₄)alkoxy, —PO₃H₂, —OPO₃H₂, —SO₃H, —OSO₃H,—NR¹⁵R¹⁶, —SR¹⁵, —CN, —NO₂, —CHO, —CO₂R¹⁵, —CONR¹⁵R¹⁶, PO₃R¹⁵R¹⁶,—OPO₃R¹⁵R¹⁶, —SO₃R¹⁵ and —OSO₃R¹⁵, wherein R¹⁵ and R¹⁶ are eachindependently selected from H and (C₁-C₄)alkyl; R¹⁰ is selected from H,CH₃, —PO₃H₂, ω-phosphonooxy(C₂-C₂₄)alkyl, and ω-carboxy(C₁-C₂₄)alkyl;and Z is —O— or —S ; with the proviso that when R³ is —PO₃R¹¹R¹², R⁴ isother than —PO₃R¹³R¹⁴.

[0047] Additionally, when R³ is —PO₃H₂, R⁴ is H, R¹⁰ is H, R¹ isn-tetradecanoyl, R² is n-octadecanoyl and R⁵ is n-hexadecanoyl, then Xis other than —O.

[0048] In the general formula above, the configuration of the 3′stereogenic centers to which the normal fatty acid acyl residues areattached is R or S, but preferably R. The stereochemistry of the carbonatoms to which R⁶ and R⁷ are attached can be R or S. All stereoisomers,enantiomers, diastereomers and mixtures thereof are considered to bewithin the scope of the present invention.

[0049] In one group of preferred embodiments, Y has the formula:

[0050] Within this group of embodiments, the acyl groups R¹, R² and R⁵will be selected such that at least two of the groups are (C₂-C₆)acyl.Further preferred are those embodiments in which the total number ofcarbon atoms in R¹, R² and R⁵ is from about 6 to about 22, morepreferably about from about 12 to about 18. In other preferredembodiments, X is O and Z is O. The subscripts n, m, p and q arepreferably integers of from 0 to 3, more preferably, 0 to 2. Of theremaining substituents, R⁶ and R⁷ are preferably H. The presentinvention further contemplates those embodiments in which the preferredsubstituents are combined in one molecule.

[0051] In another group of embodiments, R¹, R² and R⁵ are selected from(C₁₂-C₂₀)acyl with the proviso that the total number of carbon atoms inR¹, R² and R⁵ is from about 44 to about 60. More preferably, the totalnumber of carbon atoms in R¹, R² and R⁵ is from about 46 to about 52.Still further preferred are those embodiments in which X and Z are both—O—.

[0052] In another group of embodiments, Y has the formula:

[0053] As with the preferred group of embodiments provided above, inthis group the acyl groups R¹, R² and R⁵ will also be selected such thatat least two of the groups are (C₂-C₆)acyl. Further preferred are thoseembodiments in which the total number of carbon atoms in R¹, R² and R⁵isfrom about 6 to about 22, more preferably about from about 12 to about18. In other preferred embodiments, X is O. Of the remainingsubstituents, R³ is preferably phosphono (—PO₃H₂) and R⁴ is preferablyH. The present invention further contemplates those embodiments in whichvarious combinations of the preferred substituents are combined in onemolecule.

[0054] In another group of embodiments, R¹, R² and R⁵ are selected from(C₁₂-C₂₄)acyl with the proviso that the total number of carbon atoms inR¹, R² and R⁵ is from about 44 to about 60. More preferably, the totalnumber of carbon atoms in R¹, R² and R⁵ is from about 46 to about 52.Particularly preferred fatty acid groups for R¹, R² and R⁵ are normalC₁₄, C₁₆ and C₁₈ fatty acid groups. Still further preferred are thoseembodiments in which X is —O—. Similar to the shorter acyl chainembodiments provided above, R³ is preferably phosphono (—PO₃H₂) and R⁴is preferably H.

[0055] In another preferred embodiments of the present invention, Y is aradical of formula (Ib), X is O, R³is phosphono, R⁴ is H, and R¹, R² andR⁵ are selected from (C₁₂-C₂₄)acyl with the proviso that the totalnumber of carbon atoms in R¹, R² and R⁵ is from about 46 to about 52.Still further preferred are those compounds in which R² is(C₁₆-C₁₈)acyl.

[0056] The term “alkyl” by itself or as part of another substituent,means, unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e., C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl,t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl,cyclopropylmethyl, homologs and isomers of, for example, n-pentyl,n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group isone having one or more double bonds or triple bonds. Examples ofunsaturated alkyl groups include vinyl, 2-propenyl, crotyl,2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl),ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs andisomers. Typically, an alkyl group will have from 1 to 24 carbon atoms.A “lower alkyl” or is a shorter chain alkyl group, generally havingeight or fewer carbon atoms.

[0057] The terms “alkoxy”, “alkylamino” and “alkylthio” (or thioalkoxy)are used in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

[0058] The term “acyl” refers to a group derived from an organic acid byremoval of the hydroxy group. Examples of acyl groups include acetyl,propionyl, dodecanoyl, tetradecanoyl, isobutyryl, and the like.Accordingly, the term “acyl” is meant to include a group otherwisedefined as —C(O)-alkyl.

[0059] Each of the above terms (e.g., “alkyl” “acyl”) are meant toinclude both substituted and unsubstituted forms of the indicatedradical. Preferred substituents for each type of radical are providedbelow.

[0060] Substituents for the alkyl and acyl radicals can be a variety ofgroups selected from: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, -CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′C(O)NR″R′″, —NR″C(O)₂R′, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH,—NHC(NH₂)═NR′, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —CN and —NO₂ in a numberranging from zero to (2m′+1), where m′ is the total number of carbonatoms in such radical. R′, R″ and R′″ each independently refer tohydrogen and unsubstituted (C₁-C₈)alkyl. When R′ and R″ are attached tothe same nitrogen atom, they can be combined with the nitrogen atom toform a 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant toinclude 1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and the like.

[0061] The term “pharmaceutically acceptable salts” is meant to includesalts of the active compounds which are prepared with relativelynontoxic acids or bases, depending on the particular substituents foundon the compounds described herein. When compounds of the presentinvention contain relatively acidic functionalities, base addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfiric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, for example, Berge, S. M., et al, “Pharmaceutical Salts,”Journal of Pharmaceutical Science, 66, 1-19, 1977). Certain specificcompounds of the present invention contain both basic and acidicfunctionalities that allow the compounds to be converted into eitherbase or acid addition salts.

[0062] The neutral forms of the compounds may be regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents, but otherwise the salts are equivalentto the parent form of the compound for the purposes of the presentinvention.

[0063] In addition to salt forms, the present invention providescompounds which are in a prodrug form. Prodrugs of the compoundsdescribed herein are those compounds that readily undergo chemicalchanges under physiological conditions to provide the compounds of thepresent invention. Additionally, prodrugs can be converted to thecompounds of the present invention by chemical or biochemical methods inan ex vivo environment. For example, prodrugs can be slowly converted tothe compounds of the present invention when placed in a transdermalpatch reservoir with a suitable enzyme or chemical reagent.

[0064] Certain compounds of the present invention can exist inunsolvated forms as well as solvated forms, including hydrated forms. Ingeneral, the solvated forms are equivalent to unsolvated forms and areintended to be encompassed within the scope of the present invention.Certain compounds of the present invention may exist in multiplecrystalline or amorphous forms. In general, all physical forms areequivalent for the uses contemplated by the present invention and areintended to be within the scope of the present invention.

[0065] Certain compounds of the present invention possess asymmetriccarbon atoms (optical centers) or double bonds; the racemates,diastereomers, geometric isomers and individual isomers are all intendedto be encompassed within the scope of the present invention.

[0066] The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-¹²⁵ (125I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

[0067] The mono- and disaccharide compounds can be prepared by anysuitable means, many of which have been described. For example, certaincompounds useful in the present invention are described in co-pendingapplications Ser. Nos. 08/853,826, now U.S. Pat. No. 6,113,918;09/439,839 (filed Nov. 12, 1999), now U.S. Pat. No. 6,303,347; and inPCT/US98/09385 (WO 98/50300, Oct. 12, 1998) the disclosures of which areincorporated herein by reference in their entireties. Other compoundscan be prepared in a manner similar to that described for RC-552 (L34)in U.S. Pat. No. 6,013,640. Still other compounds can be prepared usingmethods outlined in Johnson, et al., J. Med. Chem. 42:4640-4649 (1999),Johnson, et al., Bioorg. Med. Chem. Lett. 9:2273-2278 (1999), andPCT/US98/50399 (WO 98/50399, Nov. 12, 1998). Still other compounds canbe prepared according to, for example, U.S. Pat. Nos. 4,436,727;4,877,611; 4,866,034; 4,912,094; and 4,987,237. In general, thesynthetic methods described in the above-noted references and othersynthetic methods otherwise familiar in the art are broadly applicableto the preparation these compounds. For example, in making compoundshaving different acyl groups and substitutions, one of skill in the artwill appreciate that the convergent methods described therein can bemodified to use alternate acylating agents, or can be initiated withcommercially available materials having appropriate acyl groupsattached.

[0068] Illustrative Pharmaceutical Compositions and Their Delivery

[0069] In another embodiment, the present invention concernspharmaceutical compositions comprising one or more of the mono- anddisaccharide compounds disclosed herein in pharmaceutically-acceptablecarriers/excipients for administration to a cell, tissue, animal orplant, either alone, or in combination with one or more other modalitiesof therapy. In a preferred embodiment, the pharmaceutical compositionsare formulated in the absence of exogenous antigen, i.e., are used inmonotherapeutic applications. For many such embodiments, thepharmaceutical compositions of the invention will comprise one or moreof the monosaccharide compounds described herein.

[0070] Illustrative carriers for use in formulating the pharmaceuticalcompositions include, for example, oil-in-water or water-in-oilemulsions, aqueous compositions with or without inclusion of organicco-solvents suitable for intravenous (IV) use, liposomes orsurfactant-containing vesicles, microspheres, microbeads and microsomes,powders, tablets, capsules, suppositories, aqueous suspensions,aerosols, and other carriers apparent to one of ordinary skill in theart.

[0071] In certain embodiments, the pharmaceutical compositions willcomprise one or more buffers (e.g., neutral buffered saline or phosphatebuffered saline), carbohydrates (e.g., glucose, mannose, sucrose ordextrans), mannitol, proteins, polypeptides or amino acids such asglycine, antioxidants, bacteriostats, chelating agents such as EDTA orglutathione, adjuvants (e.g., aluminum hydroxide), solutes that renderthe formulation isotonic, hypotonic or weakly hypertonic with the bloodof a recipient, suspending agents, thickening agents and/orpreservatives.

[0072] For certain applications, aqueous formulations will be preferred,particularly those comprising an effective amount of one or moresurfactants. For example, the composition can be in the form of amicellar dispersion comprising at least one suitable surfactant, e.g., aphospholipid surfactant. Illustrative examples of phospholipids includediacyl phosphatidyl glycerols, such as dimyristoyl phosphatidyl glycerol(DPMG), dipalmitoyl phosphatidyl glycerol (DPPG), and distearoylphosphatidyl glycerol (DSPG), diacyl phosphatidyl cholines, such asdimyristoyl phosphatidylcholine (DPMC), dipalmitoyl phosphatidylcholine(DPPC), and distearoyl phosphatidylcholine (DSPC); diacyl phosphatidicacids, such as dimyristoyl phosphatidic acid (DPMA), dipahnitoylphosphatidic acid (DPPA), and distearoyl phosphatidic acid (DSPA); anddiacyl phosphatidyl ethanolamines such as dimyristoyl phosphatidylethanolamine (DPME), dipalmitoyl phosphatidyl ethanolamine (DPPE) anddistearoyl phosphatidyl ethanolamine (DSPE). Typically, asurfactant:mono-/disaccharide molar ratio in an aqueous formulation willbe from about 10:1 to about 1 :10, more typically from about 5:1 toabout 1:5, however any effective amount of surfactant may be used in anaqueous formulation to best suit the specific objectives of interest.

[0073] The compounds and pharmaceutical compositions of the inventioncan be formulated for essentially any route of administration, e.g.,injection, inhalation by oral or intranasal routes, rectal, vaginal orintratracheal instillation, ingestion, or transdermal or transmucosalroutes, and the like. In this way, the therapeutic effects attainable bythe methods and compositions of the invention can be, for example,systemic, local, tissue-specific, etc., depending of the specific needsof a given application of the invention.

[0074] Illustrative formulations can be prepared and administeredparenterally, i.e., intraperitoneally, subcutaneously, intramuscularlyor intravenously. One illustrative example of a carrier for intravenoususe includes a mixture of 10% USP ethanol, 40% USP propylene glycol orpolyethylene glycol 600 and the balance USP Water for Injection (WFI).Other illustrative carriers include 10% USP ethanol and USP WFI;0.01-0.1% triethanolamine in USP WFI; or 0.01-0.2% dipalmitoyldiphosphatidylcholine in USP WFI; and 1-10% squalene or parenteralvegetable oil-in-water emulsion. Pharmaceutically acceptable parenteralsolvents will generally be selected such that they provide a solution ordispersion which may be filtered through a 0.22 micron filter withoutremoving the active ingredient.

[0075] Illustrative examples of carriers for subcutaneous orintramuscular use include phosphate buffered saline (PBS) solution, 5%dextrose in WFI and 0.01-0.1% triethanolamine in 5% dextrose or 0.9%sodium chloride in USP WFI, or a 1 to 2 or 1 to 4 mixture of 10% USPethanol, 40% propylene glycol and the balance an acceptable isotonicsolution such as 5% dextrose or 0.9% sodium chloride; or 0.01-0.2%dipalmitoyl diphosphatidylcholine in USP WFI and 1 to 10% squalene orparenteral vegetable oil-in-water emulsions.

[0076] Examples of carriers for administration via mucosal surfacesdepend upon the particular route, e.g., oral, sublingual, intranasal,etc. When administered orally, illustrative examples includepharmaceutical grades of mannitol, starch, lactose, magnesium stearate,sodium saccharide, cellulose, magnesium carbonate and the like, withmannitol being preferred. When administered intranasally, illustrativeexamples include polyethylene glycol, phospholipids, glycols andglycolipids, sucrose, and/or methylcellulose, powder suspensions with orwithout bulking agents such as lactose and preservatives such asbenzalkonium chloride, EDTA. In a particularly illustrative embodiment,the phospholipid 1,2 dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) isused as an isotonic aqueous carrier at about 0.01-0.2% for intranasaladministration of the compound of the subject invention at aconcentration of about 0.1 to 3.0 mg/ml.

[0077] When administered by inhalation, illustrative carriers includepolyethylene glycol or glycols, DPPC, methylcellulose, powdereddispersing agents, and preservatives, with polyethylene glycols and DPPCbeing preferred. In many instances, it will be preferred that the mono-or disaccharide compounds be in a nebulized form when administration byinhalation. Illustratively, delivery may be by use of a single-usedelivery device, a mist nebulizer, a breath-activated powder inhaler, anaerosol metered-dose inhaler (MDI) or any other of the numerousnebulizer delivery devices available in the art. Additionally, misttents or direct administration through endotracheal tubes may also beused. Delivery via an intratracheal or nasopharyngeal mode will beefficacious for certain indications.

[0078] One skilled in this art will recognize that the above descriptionis illustrative rather than exhaustive. Indeed, many additionalformulations techniques and pharmaceutically-acceptable excipients andcarrier solutions are well-known to those skilled in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens.

[0079] The compounds can be evaluated in a variety of assay formats toidentify and select those having the characteristics best suited for agiven application of the invention. For example, animal models can beused for identifying and evaluating cytokine release profiles intosystemic circulation following administration of a mono- and/ordisaccharide compound. In addition, various in vitro and in vivo modelsexist for examining changes in one or more aspects of an immune responseto different antigenic components in order to identify compounds bestsuited for eliciting a specific immune response of interest. Forexample, a compound can be contacted with target cells, such asmacrophages, dendritic cells or Langerhans cells in vitro, andelaborated cytokines can be measured. In addition, gene expressionarrays can be used to identify specific pathways activated or inhibitedby a particular mono- or disaccharide of interest.

[0080] It will be understood that, if desired, the compounds disclosedherein may be administered in combination with other therapeuticmodalities, such as antimicrobial, antiviral and antifungal compounds ortherapies, various DNA-based therapeutics, RNA-based therapeutics,polypeptide-based therapeutics and/or with other immunoeffectors. Infact, essentially any other component may also be included, given thatthe additional component(s) do not cause a significant adverse effectupon contact with the target cells or host tissues. The compositions maythus be delivered along with various other agents as required or desiredfor the specific embodiment(s) of the invention being implemented.

[0081] Illustratively, the pharmaceutical compositions of the inventioncan include, or be used in conjunction with, DNA encoding one or moretherapeutic proteins, antisense RNAs, ribozymes or the like. The DNA maybe present within any of a variety of delivery systems known to those ofordinary skill in the art, including nucleic acid expression systems,bacteria and viral expression systems. Numerous gene delivery techniquesare well known in the art, such as those described by Rolland, Crit.Rev. Therap. Drug Carrier Systems 15:143-198, 1998, and references citedtherein. Appropriate nucleic acid expression systems contain thenecessary DNA sequences for expression in the patient (such as asuitable promoter and terminating signal). In a preferred embodiment,the DNA may be introduced using a viral expression system (e.g.,vaccinia or other pox virus, retrovirus, or adenovirus), which mayinvolve the use of a non-pathogenic (defective), replication competentvirus. Suitable systems are disclosed, for example, in Fisher-Hoch etal., Proc. Natl. Acad. Sci. USA 86:317-321, 1989; Flexner et al., Ann.N.Y. Acad. Sci. 569:86 103, 1989; Flexner et al., Vaccine 8:17 21, 1990;U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S.Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner,Biotechniques 6:616 627, 1988; Rosenfeld et al., Science 252:431 434,1991; Kolls et al., Proc. Natl. Acad. Sci. USA 91:215 219, 1994; KassEisler et al., Proc. Natl. Acad. Sci. USA 90:11498 11502, 1993; Guzmanet al., Circulation 88:2838 2848, 1993; and Guzman et al., Cir. Res.73:1202 1207, 1993. Techniques for incorporating DNA into suchexpression systems are well known to those of ordinary skill in the art.

[0082] The DNA may also be “naked,” as described, for example, in Ulmeret al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science259:1691-1692, 1993. The uptake of naked DNA may be increased by coatingthe DNA onto biodegradable beads, which are efficiently transported intothe cells. It will be apparent that a pharmaceutical composition of theinvention may comprise both a polynucleotide and a protein component.

[0083] Any of a variety of additional immunostimulants may be includedin the compositions of this invention. For example, cytokines, such asGM-CSF, interferons or interleukins to further modulate an immuneresponse of interest. For example, in certain embodiments, additionalcomponents may be included in the compositions to further enhance theinduction of high levels of Th1-type cytokines (e.g., IFN-γ, TNFα, IL-2and IL-12). Alternatively, or in addition, high levels of Th2-typecytokines (e.g., IL-4, IL-5, IL-6 and IL-10) may be desired for certaintherapeutic applications. The levels of these cytokines may be readilyassessed using standard assays. For a review of the families ofcytokines, see Mosmann and Coffman, Ann. Rev. Immunol. 7:145-173, 1989.

[0084] Illustrative compositions for use in induction of Th1-typecytokines include, for example, a combination of CpG-containingoligonucleotides (in which the CpG dinucleotide is unmethylated) asdescribed, for example, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos.6,008,200 and 5,856,462. Immunostimulatory DNA sequences are alsodescribed, for example, by Sato et al., Science 273:352, 1996. Othersuitable immunostimulants comprise saponins, such as QS21 (AquilaBiopharmaceuticals Inc., Framingham, Mass.), and related saponinderiviatives and mimetics thereof.

[0085] Other illustrative immunostimulants that can be used inconjunction with the present invention include Montanide ISA 720(Seppic, France), SAF (Chiron, Calif., United States), ISCOMS (CSL),MF-59 (Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4,available from SmithKline Beecham, Rixensart, Belgium), and Enhanzyn™immunostimulant (Corixa, Hamilton, Mont.). Polyoxyethylene etherimmunostimulants, are described in WO 99/52549A1.

[0086] General Definitions:

[0087] As used herein, “an effective amount” is that amount which showsa response over and above the vehicle or negative controls. As discussedabove, the precise dosage of the compound of the subject invention to beadministered to a patient will depend the route of administration, thepharmaceutical composition, and the patient.

[0088] The phrase “pharmaceutically acceptable” refers to molecularentities and compositions that do not produce an allergic or similaruntoward reaction when administered to a human.

[0089] As used herein, “carrier” or “excipient” includes any and allsolvents, dispersion media, vehicles, coatings, diluents, antibacterialand antifungal agents, isotonic and absorption delaying agents, buffers,carrier solutions, suspensions, colloids, and the like. The use of suchmedia and agents for pharmaceutical active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated.

EXAMPLES Example 1 Protection Against P. carinii Infection byProphylactic Administration of Monophosphoryl Lipid A

[0090] Mice were pretreated with L3T4 anti-CD4 antibody for a minimum of2 weeks (2 injections/week, 0.2 mg/injection) or until the peripheralCD4 count was reduced by at least about 50%.

[0091] An aqueous formulation was prepared containing 1 mg/ml3-O-deacylated monophosphoryl lipid A and 108 μg/ml of the surfactantDPPC in water. The formulation was administered intratracheally via asmall cannula at −24 hours and then twice a week for the remainder ofthe study. The concentrations administered are indicated below inTable 1. One million P. carinii were inoculated trans-tracheally at day0. Twice-weekly treatments were continued for 7 weeks, lungs wereremoved and impression smears made. Slides were stained with Giemsa andsilver and scored for the presence of P. carinni as follows. Score5 >100/1000x field 4 10-100/field 3 1-10/field 2 1-10/10 fields 11-10/50 fields 0 0/50 fields

[0092] The results of these experiments are summarized below in Table 1:TABLE 1 GIEMSA SILVER PLACEBO GROUP 3.3 ± 0.7 2.5 ± 0.4 25 MG/KG 3.4 ±0.5 3.3 ± 0.1 100 MG/KG 2.7 ± 0.5 3.2 ± 0.1 250 MG/KG 1.8 ± 0.8 1.6 ±0.2

[0093] This study was repeated and the following results were obtained(Table 2): TABLE 2 GIEMSA PLACEBO GROUP 3.3 ± 0.2 UNTREATED CONTROL 3.1± 0.3 100 MG/KG 1.3 ± 0.3 200 MG/KG 1.0 ± 0.3

[0094] These results demonstrate that pulmonary delivery ofmonophosphoryl lipid A promotes nonspecific resistance to infection byPneumocystis carinii in immunocompromised mice. Inhalation ofmonophosphoryl lipid A led to activation of the local (and distal)innate immune responses resulting in enhanced nonspecific protection.Monophosphoryl lipid A mediated this protection primarily throughactivation of antigen presenting cells leading to increased phagocyticactivity and the release of immuno-stimulatory cytokines. FACS analysisof cell lavaged from the lungs displayed markers for activatedneutrophils but was unremarkable for an influx of leukocytescharacteristic of a massive inflammatory response (ARDS). Analysis ofspleen cells showed negative expression of CD11b or CD69, suggestingthat the monophosphoryl lipid A formulations and the effects in thisapplication were not systemic but were confined to the lung.

Example 2 Protection Against Lethal Influenza Challenge by ProphylacticAdministration of Monophosphoryl Lipid A

[0095] A dose of 20 μg MonoPhosphoryl Lipid A (MPL) was given to groupsof female BALB/c mice by intranasal (i.n.) administration either 2 daysprior to or the day of lethal influenza challenge. All mice werechallenged with approximately 2 LD50 infectious influenza A/HK/68administered i.n. Mortality was monitored for 21 days followinginfluenza challenge. The results of these experiments is presented inFIG. 1. These data demonstrate that intranasal delivery ofmonophosphoryl lipid A promotes nonspecific resistance to infection bylethal influenza challenge in mice.

Example 3 Clinical Symptoms Following Intranasal Administrative ofL-Seryl Aminalkyl Glucosaminide Phosphates (AGPs)

[0096] A series of L-Seryl Aminoalkyl Glucosaminide Phosphate compounds(AGPs) was prepared as described in U.S. Pat. No. 6,113,918, issued Sep.5, 2000, and in U.S. patent application Ser. No. 09/439,839, filed Nov.12, 1999, now U.S. Pat. No. 6,303,347, each of which is incorporatedherein by reference in its entirety.

[0097] A dose of 20 μg of L-Seryl AGPs (RC-526, RC-554, RC-555, RC-537,RC-527, RC-538, RC-560, RC-512 and vehicle only) was given to groups offemale BALB/c mice by intranasal (i.n.) administration. During theinitial 4 days following AGP administration, the mice were monitored forthree subjective indicators of disease (i.e. disease index) includingobserving ruffled fur, hunched posture and labored breathing. Theresults of these experiments is presented in FIG. 2. These dataindicated that i.n. administration of the RC-537, RC-527, RC-538 andRC-560 induce some toxicity in mice at the given dose of 20 μg.

Example 4 Clinical Symptoms Followings Intranasal Administration ofL-Seryl Aminalkyl Glucosaminide Phosphates (AGPs) and InfluenzaChallenge

[0098] A dose of 20 μg L-Seryl AGPs (RC-526, RC-554, RC-555, RC-537,RC-527, RC-538, RC-560, RC-512 and vehicle only) was given to groups offemale BALB/c mice by intranasal (i.n.) administration 2 days prior toor the day of lethal influenza challenge. All mice were challenged withapproximately 2 LD50 infectious influenza A/HK/68 administered i.n. Thedisease index (ruffled fur, hunched posture and labored breathing) wasmonitored during days 4-19 following influenza challenge. Weight lossand mortality were monitored for 21 days following influenza challenge.The results of these experiments are presented in FIG. 3.

[0099] These data demonstrated the efficacy of AGP compounds RC-538,RC560 and RC-512 in providing substantial protection against influenzachallenge.

[0100] L-Seryl AGPs having 14 carbon fatty acid chains in the primaryfatty acid position and 6 to 14 carbon fatty acids chains in thesecondary fatty acid position, (RC-526, RC-554, RC-555, RC-537, RC-527,RC-538, RC-560, RC-512, see FIG. 10), or combinations of 6 or 10 carbonfatty acids in the three secondary fatty acid positions, (RC-570,RC-568, RC-567, RC-566, RC-565, RC-569, see FIG. 10) were tested againstMPL in the influenza challenge model as described above.

[0101] The results indicate that chain length and position have aneffect on survival and disease severity. Treatment with AGP compoundshaving secondary fatty acid chains containing 9 or more carbons providedmore protection against the influenza challenge than did those withsmaller fatty acid chains. (FIG. 11). Length of the fatty acid chainrather than the dose administered, is most influential on survival,(FIG. 12). RC-526 (6 carbon chain) showed no difference in protection upto doses of 40 μg. RC-527 (10 carbon chain) provided maximum protectionover a dose range of 2.5 μg to 40 μg. Additionally, those compoundshaving at least two 10 carbon fatty acid chains (RC-565 and RC-569) weremore protective than those having fewer. (FIG. 13).

Example 5 Comparison of RC552 and MPL Using Human Whole Blood Culturesand Mouse Splenic Cultures

[0102] This Example discloses cytokine induction by the synthetic lipidA compound RC552 as compared to the modified natural substancemonophosphoryl lipid A (MPL) using human whole blood cultures and mousesplenocyte culture.

[0103] Lipid A compounds were tested by reconstitution in 0.2%triethanolamine in sterile water for irrigation, incubated at 56° C. andsonicated for 2×10 minutes at 37° C. LPS O55B5 (Sigma-Aldrich; St Louis,Mo.) was diluted into PBS.

[0104] Compounds were added to 450 μl of human whole blood and incubatedwith agitation for 5 to 24 hours. Three donors were selected (FIGS. 4-6,donors A-C, respectively). Supernatants were collected by centrifugationand diluted to 1/2 with an equal volume of PBS. (This dilution was notconsidered a dilution factor for cytokine calculations). Cytokineelaboration was measured by ELISA (R&D Systems; Minneapolis, Minn.)using the required volume of supernatant at full strength or diluted asmuch as ten fold.

[0105] BALB/c, DBA/2 and C3H/HEJ mice were purchased from The JacksonLaboratory (Bar Harbor, Me.). Spleens were taken from the mice between2PM and 3 PM, and separate single cell suspensions were obtained foreach mouse strain. Red blood cells were lysed using Tris-ammoniumchloride solution (Sigma-Aldrich), cells were washed and counted usingTrypan Blue (Sigma-Aldrich) exclusion. One million splenocytes werecultured per well in 1.0 mL of culture medium. Splenic culture medium(SCM) was designed for 5 day or longer cultures of mouse splenocytes andconsisted of RPMI 1640 (Sigma-Aldrich) supplemented to 5% with fetalbovine serum (HyClone; Logan, Utah), 100 ug/mL Gentamicin(Sigma-Aldrich), 250 ng/mL amphothericin (In Vitrogen Life Technologies;Carlsbad, Calif.), 1×ITS (bovine insulin 500 ng/mL, human transferring500 ng/mLg, sodium selenite 250 ng/mL, Sigma), beta-mercaptoethanol 43nM (Sigma-Aldrich) purivic acid 1 mM (Sigma-Aldrich), HEPES 10 mM(Sigma-Aldrich). Data from these experiments is presented herein as FIG.8.

[0106] Using human whole blood cultures, four cytokines were measured:IL-10, MIP-1 beta, TNF alpha, and IL-8. Two donors were tested once andone donor was tested twice. It is noteworthy that the donor who wastested twice had very high background TNF alpha at one test, but verylow background TNF alpha one month later. Significant, however, is thetime span of culture. High background TNF alpha was obtained with a 5.5hour culture, and low background TNF alpha was obtained with anovernight, about 24 hour, culture. Nonetheless, even the 5.5 hour-lowbackground culture was higher (about 600 pg/ml) than obtained in someprevious cultures (518.2 pg/ml in a 5 hr test; 417 pg/ml in a 4 hr test;zero pg/ml 4 hr, low responder).

[0107] RC552 was similar to MPL for elaboration of TNF alpha in two ofthree 24 hours cultures, and one 5.5 hour culture. IL-8 induction,however, by RC552 was different than that for MPL in three of threecases. IL-8 induction by RC552 was lessened for two overnight culturescompared to MPL, but greater than MPL in one overnight culture.

[0108] For overnight cultures, IL-10 induction by RC552 was less thanthat for MPL. MIP-1 beta induction was lessened in one of 3 cases ofovernight culture.

[0109] BALB/c responses and C3H/HEJ responses were compared for MPL andRC552. C3H/HEJ mice are genetic hyporesponders to LPS due to a mutationin toll-receptor 4. In these cultures, an oligonucleotide stimulant wasused as a positive control for C3H/HEJ cultures. This oligonucleotideinduced large amounts of IL-6 in BALB/c mice (1000 pg/mL) and in C3H/HEJmice (488.5 pg/mL). Similarly, MIP-1 beta was induced in BALB/c andC3H/HEJ cultures (589 pg/mL and 554 pg/mL), as was IL-10 (342 pg/mL and609 pg/mL), and TNF alpha (204 pg/mL and 30 pg/mL) in response to 10ug/mL MPL or RC552, respectively.

[0110] Neither MPL nor RC552 induced a cytokine response using C3H/HEJsplenocytes. In BALB/c splenocyte cultures, however, IL-10, MIP-1 beta,TNF alpha and IL-6 were induced. RC552 induced less MIP-1 beta, TNFalpha and IL-6 than did MPL at the same concentrations. RC552 inducedvery little IL-10 (10.4 to 11.6 pg/mL) compared to MPL (1144.1 to176.6pg/mL).

[0111] When tested in a solution of 0.2% triethanolamine, RC552 has asimilar but not identical pro-inflammatory profile for TNF alphainduction as does MPL in two of three overnight cultures and oneshort-term culture of human whole blood. See, FIGS. 4-6 (overnightcultures for donors A-C, respectively) and FIG. 7 (short-term culturefor donor A). In addition, MIP-1 beta induction by RC552 was similar intwo of three overnight cultures. Lessened IL-10 was induced by RC552than MPL in one overnight culture. IL-8 induction was different thanthat for MPL in all cases tested.

[0112] Using receptor deficient mice, it was clear that RC552 signalsvia toll-like receptor 4. Using BALB/c mice that are lipid A responsive,RC552 induced a lessened cytokine profile at the concentrations tested.Interestingly, the concentrations tested were at the high end of a doseresponse relationship, and RC552 induced slightly greater MIP-1 beta andTNF alpha at the lower concentration (100 □g/mL) than at the higherconcentration (20 □g/mL) tested.

[0113] By comparing the human and mouse cytokine profiles, syntheticlipid A compound RC552 lessened capacity for IL-10 induction in 2 daymouse splenocyte cultures and in 1 of 3 human blood cultures overnight,when tested at high concentrations of stimulant. In general, less TNFalpha was induced in overnight human blood cultures by RC552 than MPL.About equal TNF alpha levels were induced in short term (5.5 hour)cultures of human blood by RC552 compared to MPL. Microarray data usingRNA obtained from human macrophage stimulated with RC552 and MPLindicated early (1 hour) TNF alpha RNA for both compounds, and no lateTNF alpha RNA for both compounds. RC552, however, induced very little 6hour TNF alpha as opposed to MPL which had measurable 6 hour RNA.

[0114] In a separate set of experiments anticoagulated human whole bloodwas incubated in the presence of various AGPs and the supernatants weretested for quantities of cytokines. Into sterile 1.4 mL microtubes in a96 well format (Matrix) were delivered 480 μL of human whole blood, andup to 20 μl of each AGP. Those AGPs tested included, L-Seryl AGPcompounds varying in secondary fatty acid chain length (RC-526, RC-554,RC-555, RC-537, RC-527, RC-538, RC-560, RC-512) and L-Seryl AGPs havingvarious combinations of 6 and 10 chain fatty acids (RC-570, RC-568,RC-567, RC-566, RC-565, RC-569, aminoethyl AGPs (RC-523, RC-524, RC-529,RC-577, see FIG. 10), L-serinamide AGPS (RC-522, and RC-515, see FIG.10), and Serinol AGPs, (RC-545, RC-574, RC-519, RC-541, RC-540, andRC-517 see FIG. 10), and AGPs varying in backbone length, RC-529 (2carbon linker), RC-525 (3 carbon linker), RC-557 (4 carbon linker),RC-571 (6 carbon linker).

[0115] The tubes were sealed with caps supplied by the manufacturer, andthe 96 well plate apparatus was placed on its side so that themicrotubes were horizontal, placed upon a “belly dancer” agitationplatform, and cultured at 37° C. overnight, for 22 to 28 hours. At theconclusion of culture, the tubes with holder were centrifugedmomentarily to remove blood from the caps of each tube, the tubes wereopened and 500 μl of PBS were added, tubes sealed again, and inverted tomix. The addition of 500 μL of PBS facilitated centrifugation as well asrecovery of plasma supernatants. Tubes in a plate holder were thencentrifuged for 10 minutes at 1800 RPM in an IEC Centra 8R centrifuge.Two aliquots of 310 μL were then removed from each tube and storedfrozen in a 96 well format until assayed for cytokine content. Cytokines(IFN, IL-2, IL-4, IL-5, IL-6, IL-8, MIP-1β, TNFα) were evaluated byELISA (R and D Systems) and by cytometric bead array (BD Biosciences).

[0116] For each group of AGPs tested, two blood donors were used. Donor1 and Donor 2 were not the same in all experiments reported in Tables 3and 4. Each horizontal grouping represents one experiment in which donor1 and donor 2 were the same for all tests within that group. As apositive control, separate samples of the same donor's blood werestimulated with purified phytohaemagluttinin (PHA, Sigma), LPS from Ecoli O55B5 (Sigma), E. coli DNA (Sigma) as well as MPL at doses up to 20μl. Controls were used to determine the background state of the bloodcells as well as the ability of the blood cells to be stimulated.

[0117] In general, results compare favorably with those found ininfluenza challenge model and the Listeria protection model, describedherein. AGPs that were not active in these models did not inducecytokines in human blood cultures. AGPs that were weak protectors inmice, were weak inducers of cytokines in human whole blood cultures.AGPs that were very potent in mouse protection studies also were potentinducers of human cytokines. Table 3 summarized the results forcytokines IL-6, IL-8, MIP-1β, TNFα, IL-10 and IFNγ. The maximum level ofcytokine obtained is reported. TNFα and IFNγ are induced early havepeaked before the assay is performed at 24 hours. In addition, IFNγ hasnot been found in human blood cultures in a routine fashion, onlysporadically. IFNγ induction was found in an isolated case of IFNγexpression. Background IFNγ levels for that donor were high (>200pg/mL), suggesting that an immune response was in process.

[0118] Table 4 provides the maximum levels of IL-4, IL-2 and IL-5obtained, as well as ratios of TNF to IL-10, and ratios of IL-10 to TNF.Inhibition data for IL-8, reported as a percentage, are also provided onthe right hand side of Table 4. For example, 90% inhibition wouldcalculate to a 90% reduction of cytokine levels. MIP-1β results weresimilar.

[0119] RC-526, RC-554, RC-555, RC-570, RC-568, RC-567, and RC-566exhibited little if any cytokine stimulation but when added to Ogawa P.gingivalis LPS stimulated whole blood cells they inhibited LPSproduction of cytokines 90-100%. See Table 4. Such LPS blocking would beuseful for applications where reduced LPS levels are desired. Topicalapplication of such compounds would be useful to reduce bacteria levelsprior to treatment, such as for dental surgery.

[0120] The results for RC-529 (2 carbon linker), RC-525 (3 carbonlinker), RC-557 (4 carbon linker), RC-571 (6 carbon linker) suggest thatas the aglycone carbon chain length increased, the cytokine stimulatoryeffect decreased, indicating that greatest stimulation was achieved whenthe fatty acid residues attached to the aglycone moiety were closelyadjacent to the fatty acids attached to the glucosamine moiety. TABLE 3AGP IL-6 IL-8 MIP-1β TNFα cba IL-10 IFNγ cba RC# donor 1 donor 2 donor 1donor 2 donor 1 donor 2 donor 1 donor 2 donor 1 donor 2 donor 1 donor 2570 − − − − − − + − − 568 − − − − − +/− + − − 567 − − − − − +/− + − −566 − − − − + + + − − 565 − + ++ ++ ++ +++ +/− − 569 + +/− ++ +++ ++ +++++++ + − 539 ++ + +++ ++++ +++ +++ ++++ +++ − 562 +/− − + +++ ++ ++++++ + − MPL + +/− +++ ++++ +++ +++ ++++ + − None − − − − − − − − −LPS + + ++ +++ +++ ++++ ++++ +++ − EcDNA + +/− ++ − +++ + ++++ − −PHA + + ++ +++ +++ ++++ ++++ ++++ + 526 − − − − − − − − ++ 554 − − − −+/− − − − ++ 555 − + +/− +/− +/− − − − ++ 537 + ++ ++ ++++ ++++ + ++ −++ 527 + ++ + ++++ ++++ +++ +++ − ++ 538 + ++ + ++++ ++++ +++ +++ − ++560 + ++ + ++++ ++++ ++ +++ − ++ 512 + ++ + ++++ +++ − ++ − ++ MPL +++ + +++ ++ − + − + None − − − − − − − − − LPS + +++ + +++ ++ ++ +++ −++ EcDNA + +++ + +++ +++ +++ +++ − + PHA +/− +/− +/− − +/− ++ + +++ ++523 + − +++ − + + − − − − +/− − 524 ++ + ++++ ++ + +++ − − + + +/− − 529++ + ++++ ++ + ++ − − + − +/− − 525 ++ + ++++ +++ ++ ++++ + + + + +/− −557 ++ + ++++ + ++ ++ − − + − − − 571 + − +++ − + + − − − − − − 577 ++/− +++ + + + − − − − − − MPL + + +++ + + ++ − − − − − − None − − − − −− − − − − − − LPS ++ + +++ + +++ ++++ + + + ++ +/− + EcDNA ++ ++ + +++++ ++++ − − ++ ++ − − PHA + + +++ ++ + ++ − − − − +/− ++ 522 +++ ++++++ +++ +++ +++ + + − − 515 +++ +++ ++++ ++++ +++ ++ + − − − 545 ++ +++++ +++ ++ +++ − − − − 544 +++ + ++++ +++ +++ ++ ++ − − − 519 ++ + +++++++ ++ ++ − + − − 541 +++ ++ ++++ ++ ++ ++ − − − − 540 ++ ++ ++++ ++++++ ++ + +/− − − 517 +++ +++ ++++ ++++ ++ ++ +/− +/− − − MPL + ++ ++++++ + ++ − − − − None − − − − − − − − − − LPS ++ +++ ++++ ++++ ++++ +++++++ +/− − − EcDNA ++ +++ ++++ ++++ +++ ++ ++++ + − − PHA +++ +++ ++++++++ ++++ +++ +++ − +++ ++

[0121] TABLE 4 10, 5, 2.5 ug/mL AGP TNFα/IL10 cba IL10/TNFα cba % inhib.IL-1β cba2 RC# IL-4 IL-2 IL-5 donor 1 donor 2 donor 1 donor 2 LPS IL-8donor 1 donor 2 570 − − − 100 568 − − − 96 567 − − − 100 566 − − − 93565 − − − 16 569 − − − −13 539 − − − 562 − − − MPL − − − None − − − LPS− − − EcDNA − + − PHA − − − 526 − − − 0.9 1.2 99 256 554 − − − 0.9 1.1100 545 555 − − − 0.8 1.2 99 519 537 − − − 0.8 9.7 1.3 0.3 33 8353 527 −− − 2.5 3.4 0.4 0.3 5 7102 538 − − − 2.3 4 0.5 0.3 −3 7767 560 − − − 0.85.1 1.3 0.2 −1 5833 512 − − − 3.9 0.4 −1 3230 MPL − − − 1 1 1213 None −− − 0 LPS − − − 0.6 6.3 1.7 0.2 4414 EcDNA − − − 8.7 0.1 9453 PHA − ++ −0.9 1.1 2023 523 − − − −47 524 − − − 0.2 0.3 1.8 4 − 529 − − − 0.4 6.2 −525 − − − 1.8 1.2 3.7 0.7 − 557 − − − 0.6 0.5 0.6 − 571 − − − 0.5 8.6 −577 − − − − MPL − − − None − − − 0.3 LPS − − − 0.1 3.2 2.5 EcDNA − − −0.4 8.3 3.3 PHA − ++ − 522 − − − 0.4 2.7 515 − − − 0.4 0.8 3 1.4 545 − −− 544 − − − 0.6 1.7 519 − − − 541 − − − 540 − − − 0.2 0.2 5.5 5.2 517 −− − 0.3 0.7 3.9 1.4 MPL − − − None − − − LPS − − − 1.7 0.3 0.7 4 EcDNA −− − 1.8 0.5 0.6 2.7 PHA − ++ −

Example 6 RC529 Stimulatory Capabilities Compared to MPL and RC552

[0122] This Example demonstrates that RC529 has superior immunestimulatory capabilities as compared to MPL when assessed by IL-6, IL-10and MIP-1beta elaboration from human peripheral blood mononuclear cells(PBMC). In contrast, IL-8 elaboration was similar to that of MPL.

[0123] PBMC were stored frozen until used. PBMC donor designation wasAD112. PBMC at a density of 6.26×10⁵ were plated per well in a 48 wellplate in 1.0 ml of medium. Medium consisted of RPMI-1640 plus sodiumbicarbonate, 10% fetal bovine serum, 4 mM glutamine, 100 ug/mlgentamicin and 10 mM HEPES. PBMC were cultured for 22 hours at 37° C. ina carbon dioxide incubator. Supernatants were harvested and tested byELISA (R&D Systems) for IL-6, IL-8, IL-10 and MIP-1 beta concentration.Cytokine concentration in supernatants was compared to supernatantsobtained from unstimulated PBMC cultured identically.

[0124] At the doses tested, RC529 did not achieve dose-responsiveness atthe lowest dose for IL-6 or IL-8. Compared to MPL, RC529 induced moreIL-6, IL-10 and MIP-1 beta than did MPL. A disaccharide compound, RC552was generally intermediate in stimulatory capability on a mass basis.See, FIG. 9. These data show that RC529 is a strong inducer of IL-6,IL-10 and MIP-1 beta from frozen human PBMC.

[0125] The same group of L-Seryl AGP compounds (RC-526, RC-554, RC-555,RC-537, RC-527, RC-538, RC-560, RC-512) and combined 6 and 10 chainfatty acids (RC-570, RC-568, RC-567, RC-566, RC-565, RC-569) asdescribed above were tested and gave a result similar to that describedusing whole blood culture assay as described in Example 5. Those AGPcompounds having secondary fatty acid chains of 9 carbons or more, or atleast two 10 carbon fatty acids preferentially stimulated IL-6, IL-8,IL-10, and MIP-1β.

Example 7 Cell Surface Activation Markers

[0126] This experiment describes lineage specific cell surfaceactivation markers in human PBMCs activated with LPS or selected AGPs.PBMCs were harvested from a normal donor and plated at 1×10⁷ cells/wellin a 16 wells (6 well plate) with 3 ml RPMI. LPS (10 ng/ml), RC-526 (10μg/ml) or RC-527 (10 μg/ml) were added to each of 4 wells. Cells wereharvested from each of 2 wells at 4 hr and 24 hr following activationand immediately stained for the lineage and cell surface activationmarkers.

[0127] Table 5 shows the percent expression of cell surface activationmarkers in the indicated cell lineage. Table 6 shows the meanfluorescent intensities of the indicated activation marker within eachcell subset. This table only includes the cell surface activationmarkers that showed significant changes in the expression following a 4hr or 24 hr incubation with LPS, RC-527 or RC-526. TABLE 5 4 hractivation No Activation LPS RC-527 RC526 T-cells (CD3+) CD69+ 1 5 10 1Mono/Macs (CD14+) CD69+ 2 19 22 1 CD25+ 1 1 1 1 TLR2+ 51 67 85 93B-cells (CD19+) CD69+ 4 39 56 11 CD25+ 0 0 0 0 CD86+ 3 4 8 4 CD95+ 6 6 76 NK-Cells (CD56+) CD69+ 4 34 40 4 CD3+ CD69+ 2 11 11 1 24 hr activationT-Cells (CD3+) CD69+ 3 8 10 2 Mono/Macs (CD14+) CD14+ CD69+ 5 18 22 2CD14+ CD25+ 1 90 70 1 CD14+ TLR2+ 18 80 97 79 B-Cells (CD19+) CD19+CD69+ 15 49 66 22 CD19+ CD25+ 5 35 34 9 CD19+ CD86+ 17 40 51 22 CD19+CD95+ 14 71 73 15 NK-Cells (CD56+) CD69+ 4 42 50 4 CD3+ CD69+ 2 11 16 2

[0128] TABLE 6 MFI % No RC- Expression Activation LPS 527 RC526 4 hractivation Mono/Macs (CD14+) CD11b+ 100% 2283 1650 1870 2154CD54+(ICAM-1) 100% 559 876 743 502 B-cells (CD19+) CD54+ (ICAM-1) 100%57 57 65 55 24 hr activation Mono/Macs (CD14+) CD11b+ 100% 1737 893 16111986 CD54+ (ICAM-1) 100% 1452 5290 4445 1590 B-cells (CD19+) CD54+(ICAM-1 100% 110 205 211 118

Example 9 Murine Listeria monocytogenes Challenge Model

[0129] This example provides experiments evaluating the induction ofnon-specific resistance in the murine Listeria monocytogenes challengemodel performed using various AGPs and MPL. Mice (5 per group) weretreated intravenously with the 1 μg of an AGP or MPL solublized in 0.2%TEOA. Two days later the mice were challenged intravenously with a ˜10⁵L. monocytogenes 10403 serotype (provided by M. L. Gray, Montana StateUniversity, Bozeman, Mont.). Two days after the challenge, the mice weresacrificed and the number of colony forming units (CFUs) in the spleensof individual mice were determined by plating 10-fold serial dilutionsof splenic homogenates on tryptic soy agar plates. The degree ofprotection afforded by a given AGP or MPL was calculated by subtractingthe average number of bacteria per spleen (log10 value) in the group ofmice treated with a given compound, from the average number of bacteriaper spleen (log10 value) in a control group that was “sham” treated withvehicle (0.2% TEOA) prior to challenge with L. monocytogenes.

[0130] The same group of L-Seryl AGP compounds (RC-526, RC-554, RC-555,RC-537, RC-527, RC-538, RC-560, RC-512) and combined 6 and 10 chainfatty acids (RC-570, RC-568, RC-567, RC-566, RC-565, RC-569), asdescribed above, were tested. As was seen in the influenza model, thoseAGPs having fatty acids of 9 or more carbons in the secondary positionprovided the greatest protection, see FIG. 14. Those AGPs having atleast two 10 carbon fatty acid chains in the secondary position wereonly slightly less protective than RC-527 which has three 10 carbonfatty acid chains, and were more protective than MPL, see FIG. 15.

[0131] AGPs having 10-carbon fatty acids in the secondary position fromvarious families were also tested. L-Seryl (RC-527), Pyrrolidinomethyl(RC-590), Aminoethyl (RC-524), Serinamides (RC-522), Serinols andSerinol regioisomers (RC-540, RC-541, and RC-545) and miscellaneousother (RC-547, RC-558 and RC-573) AGPs provided protection that wasequal to or greater than that provided by MPL, see FIG. 16.

[0132] AGPs varying in linker length RC-529(2 carbon linker), RC-525(3carbon linker), RC-557 (4 carbon linker), and RC-571 (6 carbon linker)again showed that the greatest protection was achieved when the fattyacid residues attached to the aglycone moiety were closely adjacent tothe fatty acid residues attached to the glucosamine moiety, see FIG. 17.

[0133] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. Although the foregoinginvention has been described in some detail by way of illustration andexample for purposes of clarity of understanding, it will be readilyapparent to those of ordinary skill in the art in light of the teachingsof this invention that certain changes and modifications may be madethereto without departing from the spirit or scope of the appendedclaims.

What is claimed is:
 1. A method for modulating the production ofcytokines in a subject in need of such modulation comprisingadministering to the subject an effective amount of one or morecompounds having the formula:

and pharmaceutically acceptable salts thereof, wherein X is a memberselected from the group consisting of —O— and —NH—; R¹ and R² are eachmembers independently selected from the group consisting of(C₂-C₂₄)acyl; R³ is a member selected from the group consisting of H and—PO₃R¹¹R¹², wherein R¹¹ and R¹² are each members independently selectedfrom the group consisting of —H and (C₁₋C₄)alkyl; R⁴ is a memberselected from the group consisting of —H, —CH₃ and —PO₃R¹³R¹⁴, whereinR¹³ and R¹⁴ are each members independently selected from the groupconsisting of —H and (C₁-C₄)alkyl; and Y is a radical selected from thegroup consisting of

wherein the subscripts n, m, p and q are each independently an integerof from 0 to 6; R⁵ is (C₂-C₂₄)acyl; R⁶ and R⁷ are members independentlyselected from the group consisting of H and CH₃; R⁸ and R⁹ are membersindependently selected from the group consisting of H, OH,(C₁-C₄)alkoxy, —PO₃H₂, —OPO₃H₂, —SO₃H, —OSO₃H, -^(NR15R16), —SR¹⁵, —CN,—NO₂, —CHO, —CO₂R¹⁵, —CONR¹⁵R¹⁶, —PO₃R¹⁵R¹⁶, —OPO₃R¹⁵R¹⁶, —SO₃R¹⁵ and—OSO₃R¹⁵ and R¹⁶ are each members independently selected from the groupconsisting of H and (C₁-C₄)alkyl; R¹⁰ is a member selected from thegroup consisting of H, CH₃, —PO₃H₂, -phosphonooxy(C₂-C₂₄)alkyl, and-carboxy(C₁-C₂₄)alkyl; and Z is —O or —S—; with the proviso that when R³is —PO₃R¹¹R¹², R⁴ is other than —PO₃R¹³R¹⁴, and with the further provisothat when R³ is —PO₃H₂, R⁴ is H, R¹⁰ is H, R¹ is n-tetradecanoyl, R² isn-octadecanoyl and R⁵ is n-hexadecanoyl, then X is other than —O—.
 2. Amethod in accordance with claim 1, wherein the compound or compounds areadministered in the form of pharmaceutically acceptable salts.
 3. Amethod in accordance with claim 1, comprising administering a prodrug orprodrugs of the compound or compounds.
 4. A method in accordance withclaim 1, wherein the compound or compounds are administered in the formof a composition further comprising one or more pharmaceuticallyacceptable carriers.
 5. A method in accordance with claim 1, wherein thecompound or compounds are administered in the form of an aqueouscomposition comprising water and one or more surfactants.
 6. A method inaccordance with claim 5, wherein said one or more surfactants areselected from the group consisting of dimyristoyl phosphatidyl glycerol(DPMG), dipalmitoyl phosphatidyl glycerol (DPPG), distearoylphosphatidyl glycerol (DSPG), dimyristoyl phosphatidylcholine (DPMC),dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine(DSPC); dimyristoyl phosphatidic acid (DPMA), dipalmitoyl phosphatidicacid (DPPA), distearoyl phosphatidic acid (DSPA); dimyristoylphosphatidyl ethanolamine (DPME), dipalmitoyl phosphatidyl ethanolamine(DPPE) and distearoyl phosphatidyl ethanolamine (DSPE).
 7. A method inaccordance with claim 5, wherein the molar ratio of said compound orcompounds to surfactant is from about 10:1 to about 1:10.
 8. A method inaccordance with claim 1, wherein at least one of said R¹, R² and R⁵ areselected from the group consisting of (C₂-C₆)acyl.
 9. A method inaccordance with claim 1, wherein at least one of said R¹, R² and R⁵ isselected from the group consisting of (C₂-C₆)acyl and the total numberof carbon atoms in R¹, R² and R⁵ is from about 6 to about
 22. 10. Amethod in accordance with claim 1, wherein at least one of said R¹, R²and R⁵ are selected from the group consisting of (C₂-C₆)acyl and thetotal number of carbon atoms in R¹, R² and R⁵ is from about 12 to about18.
 11. A method in accordance with claim 1, wherein X and Z are both—O—.
 12. A method in accordance with claim 1, wherein R¹, R² and R⁵ areeach independently selected from the group consisting of (C₁₂-C₂₄)acylwith the proviso that the total number of carbon atoms in R¹, R² and R⁵is from about 44 to about
 60. 13. A method in accordance with claim 12,wherein said total number of carbon atoms is from about 46 to about 52.14. A method in accordance with claim 12, wherein X and Z are both —O—.15. A method in accordance with claim 1, wherein at least one of saidR¹, R² and R⁵ are selected from the group consisting of (C₆-C₁₂) acyl.16. A method in accordance with claim. 1, wherein at least one of saidR¹, R² and R are selected from the group consisting of (C₆-C₁₂) acyl andthe total number of carbon atoms in R¹, R² and R⁵ is from about 18 toabout
 36. 17. A method in accordance with claim 15, wherein at least oneof said R¹, R² and R⁵ is a C₆ acyl group and at least one of said R¹, R²and R⁵ is a C₁₀ acyl group.
 18. A method in accordance with claim 1,wherein said compound or compounds is administered to said subject by aroute selected from the group consisting of parenteral, oral,intravenous, infusion, intranasal, inhalation, transdermal andtransmucosal.
 19. A method in accordance with claim 1, wherein saidcompound or compounds is administered intranasally.
 20. A method inaccordance with claim 1, wherein the production of cytokines in thesubject is enhanced.
 21. A method in accordance with claim 1, whereinthe production of cytokines is inhibited.
 22. A method in accordancewith claim 1, wherein Y is

and R⁸ is CO₂H.
 23. A method in accordance with claim 22, wherein X isO, Y is O, n, m, p and q are 0; R³ is phosphono; and R⁴, R⁶, R⁷ and R⁹are hydrogen.
 24. A method in accordance with claim 22, wherein R¹, R²and R⁵ are all C₆ acyl.
 25. A method in accordance with claim 22,wherein R¹, R² and R¹ are all C₇ acyl.
 26. A method in accordance withclaim 22, wherein R¹, R² and R⁵ are all C₈ acyl.
 27. A method inaccordance with claim 22, wherein R¹, R² and R⁵ are all C₉ acyl.
 28. Amethod in accordance with claim 22, wherein R¹, R² and R⁵ are all C₁₀acyl.
 29. A method in accordance with claim 22, wherein R¹, R² and R⁵are all C₁₁ acyl.
 30. A method in accordance with claim 22, wherein R¹,R² and R⁵ are all C₁₂ acyl.
 31. A method in accordance with claim 22,wherein R¹, R² and R⁵ are all C₁₄ acyl.
 32. A method in accordance withclaim 22, wherein at least one of R¹, R² and R⁵ is C₆ acyl and at leastone other of R¹, R² and R⁵ is C₁₀ acyl.
 33. A method in accordance withclaim 22, wherein R¹ is C₁₀ acyl and R² and R⁵ are both C₆ acyl.
 34. Amethod in accordance with claim 22, wherein R⁵ is C₁₀ acyl and R¹ and R²are both C₆ acyl.
 35. A method in accordance with claim 22, wherein R¹is C₆ acyl and R² and R⁵ are both C₁₀ acyl.